1. Field of the Invention
The present invention relates to detection/quantification of targeted nucleotide chains with specific base sequences therein and detection/quantification of multi-stranded nucleotide chains such as double-stranded nucleotide chains.
2. Related Background Art
In order to detect specific genes of organisms, in the detection of a double-stranded nucleotide hybrid of a targeted nucleotide chain and a nucleotide probe for the targeted nucleotide chain, a technology has been widely used of detecting/quantifying the double-stranded nucleotide chain in gel or solution using fluorescence dyes, such as ethidium bromide (hereinafter referred to as EB), which act on double-stranded nucleotide chains and increase their fluorescence intensity.
In recent years, while polymerase chain reaction, which replicates nucleotide chains with an enzyme, has been very commonly used to detect a very slight amount of nucleotide chains, a method has also been used which uses 2-methyl-4,6-bis(4-N,N-dimethylaminophenyl)pyrylium salt, a dye emitting fluorescence only after acting on double-stranded nucleotide chains in solution, to detect the PCR amplification products (Nucleic Acid Research, 1995, Vol. 23, No. 8 1445-1446).
Further, quite recently, a fact has been known that there exist in organisms triple-stranded and quadruple-stranded nucleotide chains having special base sequences and those nucleotide chains play an important part in the replication of genes and the lifetime of cells in organisms.
These nucleotide chains form triple-stranded chains and quadruple-stranded chains and, in each chain, 3 or 4 bases in corresponding positions form a base trio or a base quartet in a plane.
Although there have been very few studies on dyes acting on these triple- and quadruple-stranded chains, in particular on fluorescence dyes, there is found in a collection of lecture resumes II (665) for the 72th spring meeting of the Chemistry Society of Japan (1997) a description that 2-methyl-4,6-bis(4-N,N-dimethylaminophenyl)pyrylium salt acts on triple- and quadruple-stranded DNAs and emit fluorescence in a solution.
Further, besides of the above dye, there have been several dyes, including YOYO1 from Molecular Probe Co., on the market in recent years.
Among these dyes, EB is generally considered to be an intercalator which enters the space between the two base pairs of double-stranded nucleotide chains, and there is found in a literature (Nucleic Acid Symposium Series No. 29 1993 83-84) a description that 2-methyl-4,6-bis(4-N,N-dimethylaminophenyl)pyrylium salt is also an intercalator. On the other hand, YOYO1 is generally considered to be a groove binding type fluorescence dye which enters the groove of double-stranded nucleotide chains.
The method, what is called solid-phase hybridization, is widely known which forms a hybrid on a solid phase by fixing a targeted nucleotide chain, which has a specific base sequence, for use in detection of a specific gene or fixing a probe nucleotide chain having a base sequence complementary to a specific site of the base sequence of the targeted nucleotide chain on a solid phase and by reacting the same with the corresponding probe nucleotide chain or targeted nucleotide chain to detect the hybrid thereof. And there have been developed various detecting techniques.
The most typical technique is the Southern hybridization technique, which forms a hybrid on, for example, a nylon filter by transferring a nucleotide chain, which is developed according to their length utilizing gel electrophoresis, to the nylon filter, denaturing the same to be single-stranded, and allowing a probe nucleotide chain labeled with a radioisotope to act on the same, so as to detect the formed hybrid thereof by using autoradiography.
And in order to overcome the problems attendant to the use of radioisotope, an improved technique has been put to practical use which uses chemiluminescence as a detecting means.
In recent years, as means for detecting a specific gene more simply and easily, a technique has also been developed which forms a hybrid on a micro-plate by fixing a probe nucleotide chain on the micro-plate and allowing a mRNA targeted nucleotide chain to act on the same, so as to detect the formed hybrid thereof with a dye, for example, ethidium bromide (hereinafter referred to as EB), which acts specifically on double-stranded nucleotide chains and thereby increase its fluorescence intensity (published Japanese Laid-Open Application of PCT Patent Application No. 7-506482).
As described above, there have been known dyes which act on double-stranded nucleotide chains or multi-stranded nucleotide chains in solution and thereby increase their fluorescence intensity or emit fluorescence for the first time; however, what is known about them is just the behavior in solution.
Usually for the determination of the fluorescence characteristics (intensity, excitation/fluorescence spectrum, etc.) of fluorescence dyes, measurements are made for about 1 to 4 mL of sample solution with a dye dissolved therein placed in measuring glass cells, generally with a spectrofluorometer. Micro-cells of about 200 μL are also commercially available. And, lately, devices are also commercially available from several manufacturers which make measurements automatically and continuously for sample solutions placed in about 100 to 250 μL wells up to 96 of a plastic micro-plate (for example, Cyto Flour, by Nippon Perceptive Ltd.).
In any of these measurements, since measurements are made for the sample solution in solution as described above, the containers for use in the measurements are restricted, further, when making measurements using a micro-plate, if the sample is illuminated with excitation light from the solution surface side, there occurs a problem of causing diffused reflection and scattering on the solution surface, and the illumination has to be conducted from the plate back side.
Further, when the amount of the sample solution is as small as 0.5 to 5 μL, the containers for use in the measurements are limited. In addition, when trying to measure fluorescence with a microscope, there arise not only a problem of excitation direction as described above, but also a problem of permitting the sample to be dried and the fluorescence of the same to be quenched, even in a typical fluorescence dye, FITC (fluorescein isothiocyanate).